Although Ca2+ dependent myosin light chain (MLC) phosphorylation is an important step in the initiation of contraction in vascular smooth muscle, we have demonstrated that force can also be developed and maintained in the absence of MLC phosphorylation. During the current funded period, we tested the hypothesis that two Ca2+ dependent regulatory systems act in parallel to control smooth muscle contraction: one is responsible for the rapid development of force by phosphorylated crossbridges and the second controls the slow development or maintenance of force by unphosphorylated crossbridges. Our results demonstrated that smooth muscle is regulated by two Ca2+ dependent processes and suggested that these systems act in parallel. Moreover, our data indicate that the second Ca2+ dependent process may involve the thin filament protein caldesmon. This proposal is based on the hypothesis that Ca2+ and phosphorylation dependent disinhibition of caldesmon allows an intrinsic level of actin-activated myosin ATPase activity to be expressed; this expression results in force development. Coexisting and simultaneous with this process is Ca2+ dependent MLC phosphorylation which significantly increases myosin ATPase activity and the rate of force development. We will also test two alternate hypotheses: 1. caldesmon disinhibition and MLC phosphorylation occur in series; and 2. the rate of MLC phosphorylation-dephosphorylation is the primary regulator of contraction. To test our hypothesis, swine carotid arteries will be studied as isolated actomyosin, Triton X-100 detergent skinned fibers and intact tissues. With these models we will determine the Ca2+ dependence of biochemical (ATPase activity, superprecipitation and protein phosphorylation levels) and mechanical (crossbridge cycling rates and attachment, and force) parameters of contraction. The specific aims are: 1. To determine if reversal of caldesmon inhibition will initiate smooth muscle contraction by asking: Can force be developed by caldesmon disinhibition without MLC phosphorylation?; Can force be developed by MLC phosphorylation without caldesmon disinhibition?; Is there an inherent level of actin-activated myosin ATPase activity in smooth muscle?; Will increases in Ca2+ and calmodulin disinhibit caldesmon and allow expression of this activity?; Are MLC kinase and/or phosphatase activities modulated by Ca2+? 2. To determine if caldesmon inhibition can be reversed by Ca2+ dependent phosphorylation we will ask: Is caldesmon phosphorylated during a Ca2+ dependent, MLC phosphorylation independent contraction?; Is MAP kinase or Ca2+/calmodulin dependent protein kinase II responsible for caldesmon phosphorylation? 3. To determine the extent to which regulation by caldesmon plays a role in the physiological response of vascular smooth muscle we will examine: Does caldesmon disinhibition occur in response to all modes of stimulation?; Is caldesmon phosphorylation required for disinhibition in intact tissue?; and Which kinase is responsible?